Chemosphere 58 (2005) 1041–1047
www.elsevier.com/locate/chemosphere
Microbial community and biochemistry process
in autosulfurotrophic denitrifying biofilm
Albert Koenig *, Tong Zhang, Ling-Hua Liu, Herbert H.P. Fang
Department of Civil Engineering, Centre for Environmental Engineering Research,
The University of Hong Kong, Pokfulam Road, Hong Kong, China
Received 13 June 2004; received in revised form 21 September 2004; accepted 22 September 2004
Abstract
The 16S rDNA-based molecular technique was applied to analyze the microbial community of autotrophic denitrification bacteria in a biofilm developed on the surface of sulfur particles and then the biochemistry process involved in
this biofilm was discussed based on the microbial community analysis. Six key operational taxonomy units were identified, which were all unknown species belonging to a wide range of bacteria from four major subdivisions (a, b, c and d)
of the kingdom Proteobacteria and from the kingdom Chlorobia (green sulfur bacteria). One species was chemoautotrophic and related to Thiobacillus denitrificans, two species were photoautotrophic, and three were chemoheterotrophic. Contrary to expectation, T. denitrificans-like bacteria constituted only 32% of the microbial community. As
a result of the study, the entire microbiology of the autosulfurotrophic denitrification process as well as the interactions
between the different microbial groups in the biofilm may need to be reconsidered.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Autotrophic denitrification; Biofilm; Nitrogen cycle; Sulfur; Thiobacillus; Wastewater
1. Introduction
A wide variety of bacteria can use nitrate as a terminal electron acceptor. These bacteria are called nitratereducing bacteria, which includes heterotrophs using organic compounds as electron donors and autotrophs
using inorganic compounds such as sulfide, sulfur, thiosulfate or ferrous iron as electron donors. Products of
nitrate reduction include NO
2 , NO, N2O, N2, and
NHþ
.
When
N
is
produced,
the process is called
2
4
denitrification.
*
Corresponding author. Tel.: +852 2859 2655; fax: +852
2559 5337.
E-mail address: [email protected] (A. Koenig).
Biological denitrification has become an integral part
of modern wastewater treatment. For wastewater with a
low BOD5/N ratio, autotrophic denitrification is an
interesting alternative to heterotrophic denitrification.
Autotrophic denitrifying bacteria (ADB) include autohydrogenotrophic denitrifiers (Lee and Rittmann,
2000) as well as autosulfurotrophic denitrifiers, which
reduce sulfur compounds, such as elemental sulfur or
thiosulfate, to sulfate while reducing nitrate to elemental
nitrogen gas. Contrary to heterotrophic denitrification,
autotrophic denitrification eliminates the need for addition of organic carbon sources, consumes alkalinity and,
in addition, generates high concentrations of sulfate.
Autotrophic denitrification using elemental sulfur
has been investigated to remove nitrate from polluted
groundwater (Kruithof et al., 1988) and some selected
0045-6535/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.09.040
1042
A. Koenig et al. / Chemosphere 58 (2005) 1041–1047
wastewaters with high nitrate concentrations such as
nitrified leachate (Koenig and Liu, 1996, 2001, 2002).
For Thiobacillus denitrificans, the stoichiometric reaction of autotrophic denitrification using elemental sulfur
can be represented by the following equation (Batchelor
and Lawrence, 1978):
þ
NO
3 þ 1:10S þ 0:40CO2 þ 0:76H2 O þ 0:08NH4
Thiobacillus denitrificans
þ
ƒƒƒƒƒƒƒƒƒƒƒƒƒ! 0:5N2 þ 1:10SO2
4 þ 1:28H
þ 0:08C5 H7 O2 N
ð1Þ
Although autotrophic denitrification using elemental
sulfur has been extensively studied since 1978 (Batchelor
and Lawrence, 1978), knowledge about the microbial
community in the biofilm on the sulfur particle surface
is still very limited. It is generally assumed that T. denitrificans is the microbial species responsible for autotrophic denitrification (Batchelor and Lawrence, 1978).
This bacterium can reduce nitrate to nitrogen gas while
oxidising elemental sulfur or reduced sulfur compounds
to sulfate. Other autotrophic denitrifiers in addition to
T. denitrificans, such as Thiomicrospira denitrificans,
could also perform the sulfur-based autotrophic denitrification. However, no detailed analyses on the autotrophic denitrification microbial community have been
reported.
Traditionally, microbes are identified by isolating
individual cultures and examining their physiological,
biochemical and morphological characteristics. These
methods are often unreliable, because some microbes
are syntrophically associated with others and thus cannot be isolated and cultured individually (Wagner
et al., 1993). Furthermore, many microbes share similar
physiological, biochemical and morphological characteristics, and cannot be distinguished from one another
based on these characteristics. However, microbial communities may be analyzed using advanced molecular
techniques. Among them, 16S rDNA-based methods
have been extensively applied in various studies (Zhang
and Fang, 2001; Fang et al., 2004).
In this study, the 16S rDNA-based method was applied to analyze the microbial community of the ADB
in a biofilm developed on the surface of sulfur particles.
2. Materials and methods
250 ml Erlenmeyer flasks at 25 °C. Each flask contained
a total of 200 ml elemental sulfur and limestone (2.8–
5.6 mm diameter) at a volumetric ratio between 1:0 and
1:4. The batch experiments lasted about 5 h each, with
the specific denitrification rate in all batch experiments
1
amounting to approximately 0.030 mg NO
S
3 -N g
1
h , independent of the sulfur to limestone ratio. The initial cultures were prepared from sludge collected from
tidal flats of the Mai Po marshes in Hong Kong. The steps
for forming the biofilm on the sulfur particles were as follows: the reactors were inoculated with 50 ml enrichment
solution and 100 ml medium according to the enrichment
method for T. denitrificans as previously described (Koenig and Liu, 1996). Every 8 h, 120 ml solution in the reactors was decanted and 120 ml fresh medium was added.
After 10 d incubation, the medium was changed to synthetic wastewater. After a further two weeks of incubation, the denitrification rate became stable and the
batch experiments were started.
2.2. DNA extraction and amplification
The microstructure of the biofilm was examined
using scanning electron microscopy (SEM, Stereoscan
360, Cambridge, USA). Biofilm was removed from the
surface of the sulfur particles by sonification in phosphate buffer saline (130 mM NaCl, 7 mM Na2HPO4,
3 mM NaH2PO4, pH 7.4). Then the genomic DNA in
the biofilm was extracted (Zhang and Fang, 2001). For
denaturing gradient gel electrophoresis (DGGE) screening, the extracted DNA was amplified by polymerase
chain reaction (PCR) using an automated thermal cycler
(GeneAmpÒ PCR 9700, Perkin-Elmer, Foster City, CA)
as follows: an initial denaturation at 94 °C for 7 min; 35
cycles of denaturation (1 min at 92 °C), annealing (1 min
at 54 °C) and extension (1 min at 72 °C); a final extension
at 72 °C for 10 min and then stored at 4 °C. The same
procedures were also applied for DNA cloning, but only
25 cycles of denaturation, annealing and extension were
carried out. All PCR amplifications were conducted in
30 ll of a pH 8.3 buffer (Pharmacia Biotech Inc., Piscataway, NJ) containing 200 lM each of the four deoxynucleotide triphosphates, 15 mM MgCl2, 0.1 lM of
individual primers and 1U of Taq polymerase (Pharmacia Biotech Inc. Piscataway, NJ). The purity of
PCR products were checked by electrophoresis profile
using 1% agarose gel.
2.1. Reactor operation
2.3. Cloning and sequencing
The sulfur particles were obtained from batch experiments carried out to study the use of limestone for pH
control in autotrophic denitrification (Liu and Koenig,
2002). In these experiments, 150 ml of synthetic wastewater (100 mg l1 NO
1000 mg l1 NaHCO3,
3 -N,
120 mg l1 K2HPO4, 12 mg l1 NH4Cl, 2 mg l1 MgCl2 Æ
6H2O and 0.1 mg l1 FeSO4 Æ 7H2O) were incubated in
The extracted DNA was PCR-amplified using the
primer set of EUB8F and EUB1509R (Table 1) (Zhang
et al., 2003). Cloning of these fragments was conducted
using the TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA). A total of 72 colonies were selected for inoculation in a 1.0 ml LB medium containing 50 mg l1
A. Koenig et al. / Chemosphere 58 (2005) 1041–1047
1043
Table 1
DNA sequences of primers and GC-clamp
Primer
EUB8F
EUB1509R
M13F
M13R
Sequence
0
5 -AGA GTT TGA TCM TGG CTC AG
5 0 -GGT TAC CTT GTT ACG ACT T
5 0 -GTA AAA CGA CGG CCA G
5 0 -CAG GAA ACA GCT ATG AC
Specificity
Target position
Reference
Bacteria
Prokaryote
pCRÒ 2.1Vector
pCRÒ 2.1Vector
8–27
1491–1509
–
–
Lane (1991)
Lane (1991)
Invitrogen (1999)
Invitrogen (1999)
kanamycin. After 18 h incubation at 37 °C, the plasmids
were recovered. The primer set of M13F and M13R
(Table 1) was used to amplify the inserted rDNA fragments carried on the plasmid.
Judging from the DGGE profiles of the PCR products, a total of 72 clones were selected. They were composed of 12 unique DNA sequences, commonly referred
as operational taxonomy units (OTUs). Six major OTUs
were shared by three or more clones. The DNA of these
OTUs were then sequenced using an autosequencer
(ABI model 377A, Perkin-Elmer Ltd., Foster City,
CA) and dRhodamine Terminator Cycle Sequencing
FS Ready Reaction Kit (Perkin-Elmer Ltd., Foster
City, CA).
1985) for 500 replicates was performed to estimate the
confidence of the tree topologies.
2.4. Phylogenetic analysis
Fig. 1 illustrates the denitrifying biofilm formed on
the sulfur particle surface. The morphology of the bacterial cells appears to exhibit low diversity. However, further analysis using PCR-cloning method revealed that
the denitrification biofilm is more diverse than that implied by the SEM image.
Table 2 summarizes the sequence length, number of
clones and relative abundance, plus the closest species
found in the GenBank and the degree of similarity by
Blast analysis. Fig. 2 is the phylogenetic tree of the OTUs
obtained in this study and their close related species.
Each DNA sequence was compared with the reference microorganisms available in the GenBank by
BLAST search (Altschul et al., 1990). The obtained
DNA sequences and their closest 16S rDNA sequences
of reference microorganisms retrieved from the GenBank were aligned by BioEdit (Hall, 1999) to construct
phylogenetic trees using the neighbor-joining method
(Saitou and Nei, 1987) by MEGA 2.1 (Kumar et al.,
1993). Bootstrap re-sampling analysis (Felsenstein,
2.5. Accession number
The nucleotide sequence data reported in this paper
have been submitted to the GenBank, EMBL and DDBJ
databases and assigned the accession numbers
AY261821-AY261826.
3. Results and discussions
3.1. Microbial community analysis
Fig. 1. SEM image of microbial community on the surface of sulfur particle.
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A. Koenig et al. / Chemosphere 58 (2005) 1041–1047
Table 2
Six key OTUs and their closest species
OTU
Sequence
length
Closest species in GenBank
Similarity
(%)
Clone
no.
ADB-2
ADB-67
ADB-66
ADB-13
ADB-3
ADB-12
Six OTUs with the clone number of 1 or 2
334
321
399
400
305
317
Thiobacillus denitrificans
Trichlorobacter thiogenes
Uncultured bacterium PHOS-HE36
Xanthomonas maltophilia
Rhodobacter sphaeroides
Sinorhizobium sp.
95
87
97
93
94
97
23
16
12
6
4
3
8
32
22
17
8
6
4
11
72
100
Total
72 Rhodobacter sphaeroides
62
Rhodobacter azotoformans
42
ADB-3
Rhodovulum adriaticum
Rhodovulum euryhalinum
79
Rhodobacter apigmentum
80
Rhodobacter sp.TCRI2
51
ADB-12
29
Mesorhizobium tianshanense
100
Sinorhizobium sp.MSMC411
79
58 Sinorhizobium xinjiangensis
47
ADB-67
Trichlorobacter thiogenes
Desulfuromonas thiophila
44
55
Desulfuromonas palmitatis
Desulfuromonas palmitatis
73
62
Desulfuromusa bakii
51
Pelobacter acetylenicus
33
ADB-13
91
Xanthomonas maltophilia
93
Xanthomonas populi
97 Xanthomonas vesicatoria
66
Thiobacillus baregensis
33
Bordetella hinzii
49
ADB-2
99
Thiobacillus denitrificans
96
Thiobacillus thioparus
80
ADB-66
100
uncultured bacterium PHOS-HE36
99 Chlorobium vibrioforme
41
Pelodictyon luteolum
Prosthecochloris aestuarii
99
Chlorobium limicola
40
100 Chlorobium phaeobacteroides
Desulfovibrio gabonensis
Desulfurococcus mobilis
94
Abundance
(%)
Photosynthesis
Bacteria in α Proteobacteria
Rhizobiaceae
group in α Proteobacteria
Desulfuromonas
group in δ Proteobacteria
γ -Proteobacteria
δ -Proteobacteria
Green sulfur
bacteria (GSB)
0.05
Fig. 2. Phylogenetic tree of six key OTUs in autosulfurotrophic denitrification biofilm and their close relatives based on 310
nucleotides in 16S rDNA sequence. The tree based on Jukes-Cantor distance was constructed using the neighbor-joining algorithm
with 500 bootstrappings. Desulfurococcus mobilis in the Archaea domain was selected as the outgroup species. The scale bar represents
0.05 substitutions per nucleotide position. Numbers at the nodes are the bootstrap values. (j) OTUs obtained in this study.
A. Koenig et al. / Chemosphere 58 (2005) 1041–1047
Table 2 shows that the OTU ADB-2 (AY261822,
comprising 23 clones) is most closely related to the
known species T. denitrificans of b-Proteobacteria with
95% similarity. T. denitrificans is a well-known autotrophic denitrifier using elemental sulfur as the energy
source producing SO2
(Batchelor and Lawrence,
4
1978; Koenig and Liu, 1996). Fig. 2 illustrates that
ADB-2 formed a cluster, at a node bootstrap value of
96%, with two species of genus Thiobacillus, i.e. T. denitrificans and T. thioparus, but distantly related to another Thiobacillus species, T. baregensis. It is thus
likely that ADB-2, comprising 32% of total clones,
might be an uncultured new species with the physiological characteristics of T. denitrificans.
OTU ADB-67 (AY261824, comprising 16 clones) is
most closely (87% similarity) related to Trichlorobacter
thiogenes, the only species in a new genus in the Desulfuromonas group of d-Proteobacteria. Due to the lack of
similarity to any known sequences or species, ADB-67
cannot be assigned to any known taxonomic group in
the Desulfuromonas group. It may constitute a special
species, quite different from other species in the Desulfuromonas group, which reduces sulfur to hydrogen sulfide
using acetate, propionate and ethanol as the electron
donor, but never reduces sulfate or other oxoanions of
sulfur (Holt et al., 1994).
OTU ADB-66 (AY261826, comprising 12 clones) is
most closely related to an uncultured photosynthetic
bacterium PHOS-HE36 (97% similarity) previously
found in a denitrification community (Dabert et al.,
2001). Fig. 2 also shows that OTU ADB-66 clusters with
PHOS-HE36 at the bootstrap value of 100% and groups
together with the representative species of the green sulfur bacteria (GSB), such as Chlorobium phaeobacteroides, Pelodictyon luteolum, Pelodictyon aestuarii,
Chlorobium vibrioforme, and Chlorobium limicola. Thus
ADB-66 is most likely a member of the kingdom GSB,
which is photoautotrophic, using CO2 as sole carbon
source but also able to use acetate as a carbon source,
and capable of oxidizing H2S into elemental S or further
to sulfate. It is frequently found in hydrogen sulfide containing mud and water of freshwater, brackish water,
and marine environments (Holt et al., 1994). Sometimes
thiosulfate or hydrogen can also be used as electron
donor, besides elemental sulfur.
OTU ADB-13 (AY261823, comprising six clones) is
most closely related to Xanthomonas maltophilia at the
similarity of 93%. Fig. 2 illustrates that they cluster together at the node bootstrap value of 91% and cluster
with Xanthomonas populi and Xanthomonas vesicatoria
with a 93% node bootstrap value. It is thus likely that
ADB-6 is a member of Xanthomonas, which is a heterotrophic denitrifier, but may also be a parasite of GSB
(Holt et al., 1994).
OTU ADB-3 (AY261825, comprising four clones) is
closely related (94% similarity) to Rhodobacter sphaero-
1045
ides. This OTU likely belongs to the photosynthetic bacteria in a-Proteobacteria as it clusters with the reference
photosynthetic bacterial species as illustrated in Fig. 2.
Rhodobacter species could be photoautotroph (anaerobic), photoheterotroph (anaerobic/light) or chemoheterotroph (aerobic) (Holt et al., 1994).
Table 2 shows that OTU ADB-12 (AY261821, comprising three clones) is closely related (97% similarity)
to Sinorhizobium sp. and might be assigned to the Rhizobiaceae group in a-Proteobacteria as illustrated in Fig. 2.
Thus OTU ADB-12 might comprise heterotrophic bacteria using the dead and lysed bacterial cells as their
energy source.
3.2. Diversity of the biofilm microbial community
Based on the wastewater composition, autosulfurotrophic denitrifiers, such as T. denitrificans, would be
expected to be predominant in the biofilm. Yet, the
T. denitrificans-like ADB-2 represented only 32% of
the microbial community. The remaining microbes in
the biofilm consisted of a wide range of bacteria from
four major subdivisions (a, b, c, and d) of the kingdom
Proteobacteria and from the kingdom Chlorobia (the
GSB). One OTU was chemoautotrophic, two were phototrophic (at anaerobic condition), and three were
heterotrophic. The dead and lysed cells of the autotrophs
were likely the carbon source for the heterotrophic bacteria. This may indicate that even so-called pure autotrophic microbial communities will always contain a
certain proportion of heterotrophs, feeding on the organic matter (autotrophs, intermediate and degradation
products) produced. Table 3 indicates the possible biochemical reactions taking place in the autosulfurotrophic
denitrification biofilm based on the classification of the
key OTUs and the relationships between them.
The proposed relationships among the major species
and their possible distribution in the autosulfurotrophic
denitrification biofilm on the sulfur particle are schematically illustrated in Fig. 3. As stated by Batchelor and
Lawrence (1978), the microbial activity in the biofilm depends mainly on two steps, (i) sulfur (electron donor)
must be transported from the surface of the sulfur particle
through the biofilm while being microbially oxidized, and
(ii) nitrate (electron acceptor) must be transported from
the bulk liquid through the biofilm while being microbially reduced. It is suggested that, initially, ADB-2 forms
a single layer biofilm on the sulfur surface. With the thickness of the biofilm increasing, the ADB-2 cells next to the
sulfur surface are not able to obtain sufficient NO
3 for
denitrification, become inactive and lyse. The organic
matter from the lysed cells provides the food for the sulfur
reducing chemoheterotrophs ADB-67 which, in the absence of NO
3 , reduce S to H2S and inhabit the biofilm
bottom layer. Meanwhile, the H2S formed by ADB-67
is utilized by the autotrophs ADB-3 and ADB-66, which
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A. Koenig et al. / Chemosphere 58 (2005) 1041–1047
Table 3
Possible biochemical reactions of the major OTUs in this study
Trophic type
OTU
Electron donor
Electron acceptor
Oxic state
Autotrophs (54% of the total)
ADB-2
ADB-66
ADB-3
S!
H2S ! S ! SO2
4
H2S ! S ! SO2
4
NO
3
!
! N2
CO2 + H2O ! OM
CO2 + H2O ! OM
Anaerobic
Anaerobic
Facultative
Heterotrophs (35% of the total)
ADB-67
ADB-13
ADB-12
OM ! CO2 + H2O
OM ! CO2 + H2O
OM ! CO2 + H2O
SO2
4 ! S ! H2S
NO
3 ! NO2
NO3 ! NO
2 ! N2
Anaerobic
Facultative
Facultative
SO2
4
NO
2
Note: OM = organic matter (CnHaOb). The electron donors or acceptors are in bold.
Fig. 3. Suggested relationships among the major OTUs and
their possible distribution in the autosulfurotrophic denitrification biofilm as a function of substrate concentration and depth
(modified from the schematic of sulfur–biofilm system with
zero-order nitrate rate limitation by Batchelor and Lawrence,
1978).
probably occupy the middle layer of the biofilm, to form
S or further SO
4 . The chemoheterotrophs ADB-12 and
ADB-13 also depend on organic matter from lysed cells,
but appear to inhabit mostly the middle and outer layer
of the biofilm, where both organic matter and NO
3 are
available. As the biofilm grows over a longer duration
of time, its makeup could change from a single layer to
two layers or even three layers, depending on the (i) transport and reaction rate limitations of electron donor/
acceptor, and, (ii) the varying environmental conditions
within the biofilm. This could explain the presence of so
many microbial groups. But their proposed distribution
in the biofilm could not be verified as no further investigations were carried out to determine the layered structure and microbial composition of the biofilm. Evidence
of incomplete penetration of nitrate in the biofilm and
hence for the formation of two layers was demonstrated
in sulfur-packed bed reactors, when autotrophic denitrification rates followed a half-order kinetic model (Koenig
and Liu, 2001). The theoretical basis for this model was
first established by Harremoes (1978), as a consequence
of partly penetrated pores in biofilms.
The results demonstrate, however, that the biofilm has
a much more complex microbial community and metabolic diversity than originally thought. Although the
composition of the microbial community might reflect
the source of the bacteria, it is believed that the welldefined experimental conditions must have led to the
development of a stable, specialized community of ADBs.
One has to be cautious in interpreting results of this
study because of certain intrinsic limitations of the
molecular technique applied. Using this method, the
microbial DNA has to be extracted, followed by PCR
amplification and cloning. The accuracy of this method
depends on the reliability of each individual step. It is
possible that the DNA of some species could not be effectively extracted because they are difficult to lyse even by
vigorous bead beating (Curtis and Craine, 1998). Also,
some DNA fragments could be amplified more efficiently
than others, so that the former would be overrepresented
in the PCR products (Muyzer et al., 1993).
The detailed biochemical interactions between the
different microbial groups discovered could not yet be
fully established, qualitatively or quantitatively, and
merit further investigation, including the determination
of the environmental conditions within the biofilm as a
function of distance from the sulfur surface (concentrations of S and N compounds, pH, oxidation reduction
potential, etc.).
4. Conclusion
The results obtained by application of the 16S
rDNA-based molecular technique indicate that the
A. Koenig et al. / Chemosphere 58 (2005) 1041–1047
microbial community of an autotrophic denitrification
biofilm developed on the surface of sulfur particles was
much more complex than previously thought. Contrary
to expectation, T. denitrificans-like bacteria constituted
only 32% of the microbial community. Other populations included at least two autotrophs and three
heterotrophs. A tentative scheme of the interactions
among the identified microbial groups was proposed.
As a result of this study, the entire microbiology of the
autosulfurotrophic denitrification process may need to
be reconsidered.
Acknowledgement
This research has been supported by the Research
Grants Council of the Hong Kong Special Administrative Region, China (Project No.: HKU7006/98E).
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